Open Access2008Joneset al.Volume 9, Issue 7, Article R114ResearchThe transcriptional program underlying the physiology of clostridial sporulationShawn W Jones*†‡, Carlos J Paredes*§, Bryan Tracy*, Nathan Cheng*¶, Ryan Sillers*, Ryan S Senger†‡ and Eleftherios T Papoutsakis†‡

Addresses: *Department of Chemical and Biological Engineering, Northwestern University, Sheridan Road, Evanston, IL 60208-3120, USA. †Department of Chemical Engineering, University of Delaware, Academy Street, Newark, DE 19716, USA. ‡Delaware Biotechnology Institute, University of Delaware, Innovation Way, Newark, DE 19711, USA. §Current address: Cobalt Biofuels, Clyde Avenue, Mountain View, CA 94043, USA. ¶Current address: The Zitter Group, New Montgomery Street, San Francisco, CA 94105, USA.

Correspondence: Eleftherios T Papoutsakis. Email: epaps@udel.edu

© 2008 Jones et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Clostridial sporulation<p>A detailed microarray analysis of transcription during sporulation of the strict anaerobe and endospore former <it>Clostridium aceto-butylicum</it> is presented.</p>

Abstract

Background: Clostridia are ancient soil organisms of major importance to human and animalhealth and physiology, cellulose degradation, and the production of biofuels from renewableresources. Elucidation of their sporulation program is critical for understanding importantclostridial programs pertaining to their physiology and their industrial or environmentalapplications.

Results: Using a sensitive DNA-microarray platform and 25 sampling timepoints, we reveal thegenome-scale transcriptional basis of the Clostridium acetobutylicum sporulation program carrieddeep into stationary phase. A significant fraction of the genes displayed temporal expression in sixdistinct clusters of expression, which were analyzed with assistance from ontological classificationsin order to illuminate all known physiological observations and differentiation stages of thisindustrial organism. The dynamic orchestration of all known sporulation sigma factors wasinvestigated, whereby in addition to their transcriptional profiles, both in terms of intensity anddifferential expression, their activity was assessed by the average transcriptional patterns ofputative canonical genes of their regulon. All sigma factors of unknown function were investigatedby combining transcriptional data with predicted promoter binding motifs and antisense-RNAdownregulation to provide a preliminary assessment of their roles in sporulation. Downregulationof two of these sigma factors, CAC1766 and CAP0167, affected the developmental process ofsporulation and are apparently novel sporulation-related sigma factors.

Conclusion: This is the first detailed roadmap of clostridial sporulation, the most detailedtranscriptional study ever reported for a strict anaerobe and endospore former, and the firstreported holistic effort to illuminate cellular physiology and differentiation of a lesser knownorganism.

Published: 16 July 2008

Genome Biology 2008, 9:R114 (doi:10.1186/gb-2008-9-7-r114)

Received: 5 March 2008Revised: 6 June 2008Accepted: 16 July 2008

The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2008/9/7/R114

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.2

BackgroundClostridia are of major importance to human and animalhealth and physiology, cellulose degradation, bioremedia-tion, and for the production of biofuels and chemicals fromrenewable resources [1]. These obligate anaerobic, Gram-positive, endospore-forming firmicutes include several majorhuman and animal pathogens, such as C. botulinum, C. perf-ringens, C. difficile, and C. tetani, the cellulolytic C. thermo-cellum and C. phytofermentans, several ethanologenic [2],and many solventogenic (butanol, acetone and ethanol) spe-cies [3]. Their sporulation/differentiation program is criticalfor understanding important cellular functions or programs,yet it remains largely unknown. We have recently examinedthe similarity of the clostridia and bacilli sporulation pro-grams using information from sequenced clostridial genomes[1]. We concluded that, based on genomic information alone,the two programs are substantially different, reflecting thedifferent evolutionary age and roles of these two genera. Wehave also argued that C. acetobutylicum is a good modelorganism for all clostridia [1]. Transcriptional or functionalgenomic information is, however, necessary for detailingthese differences and for understanding clostridial differenti-ation and physiology. Key issues awaiting resolution include:the identification of the mid to late sigma and sporulation fac-tors and their regulons; the orchestration and timing of theiraction; the set of genes employed by the cells in the mid andlate stages of spore maturation; identification of candidatehistidine kinases that might be capable of phosphorylatingthe master regulator (Spo0A) of sporulation; and some func-tional assessment of the roles of several sigma factors ofunknown function encoded by the C. acetobutylicumgenome. Furthermore, an understanding of the transcrip-tional basis of the complex physiology of this organism will goa long way to improve our ability to metabolically engineer,for practical applications, its complex sporulation and meta-bolic programs. Such information generates tremendous newopportunities for further exploration of this complex anaer-obe and its clostridial relatives, and constitutes a firm basisfor future detailed genetic and functional studies.

Using a limited in scope and resolution transcriptional study,we have previously shown that it is possible to use DNA-microarray-based transcriptional analysis to generate valua-ble functional information related to stress response [4,5],initiation of sporulation [6] and the early sporulation pro-gram of C. acetobutylicum [7]. In order to be able to accu-rately study the transcriptional orchestration underlying thecomplete sporulation program of the cells, it was necessary todevelop a more sensitive and accurate microarray platform, abetter mRNA isolation protocol (in order to isolate RNA fromthe mid and late stationary phases), as well as to use a muchhigher frequency of observation and sampling. We also aimedto employ more sophisticated bioinformatic tools in order toglobally interrogate any desirable cellular program and relateit to the characteristic phenotypic metabolism and sporula-tion of this organism. The results of this extensive study are

presented here as a single, undivided story, which offersunprecedented insights and a tremendous wealth of informa-tion for further explorations. Furthermore, it serves as a par-adigm of what can be effectively accomplished with the nowhighly accurate DNA-microarray analysis in generating arobust transcriptional roadmap and in illuminating the phys-iology of a lesser understood organism.

Results and discussionMetabolism and differentiation of C. acetobutylicum: identification of a new cell type?We aimed to relate the metabolic and morphological charac-teristics of the cells in a typical batch culture, whereby cellsunderwent a full differentiation program, to the transcrip-tional profile of the cell population [8]. The metabolism ofsolventogenic clostridia is characterized by an initial acidog-enic phase followed by acid re-assimilation and solvent pro-duction [7]. As shown in Figure 1a, the peak of butyrateconcentration, around 16 hours after the start of the culture,coincided with the initiation of butanol production. Aroundthis time, the culture transitioned from exponential growth tostationary phase and initiated solventogenesis and sporula-tion. This period is called the transitional phase and is indi-cated by the gray bar in Figure 1a and all following figures.The butanol concentration increased to over 150 mM untilhour 45, after which no substantial change in solvent or acidconcentration took place. Nevertheless, cells continued todisplay morphological changes well past hour 60. Solven-togenic clostridia display a series of morphological forms overthis differentiation program: vegetative, clostridial, fore-spore, endospore, and free-spore forms [9]. In addition tophase-contrast microscopy, we found that by using Syto-9 (agreen dye assumed to stain live cells) and propidium iodide(PI; a red dye assumed to stain dead cells) [10] we couldmicroscopically distinguish these morphologies and identifynew cell subtypes. Staining by these two dyes did not followtypical expectations. During exponential growth, vegetativecells, characterized by a thin-rod morphology, were visiblymotile under the microscope, which is consistent with thefinding that chemotaxis and motility genes were highlyexpressed during this time [7]. When double stained withSyto-9 and PI dyes, these vegetative cells took on a predomi-nantly red color, indicating the uptake of more PI than Syto-9 (Figure 1b, I, II). At the onset of butanol production, swol-len, cigar-shaped clostridial-form cells began to appear (Fig-ure 1b, III). These clostridial forms (confirmed by phase-contrast microscopy; data not shown), generally assumed tobe the cells that produce solvents [8], were far less motilethan exponential-phase cells and stained almost equally withboth dyes, taking on an orange color. Clostridial forms per-sisted until solvent production decreased, after which fore-spore forms (cells with one end swollen, which is indicative ofa spore forming) and endospore forms (cells with the middleswollen, which is indicative of a developing spore) becamevisible [9]. These cells stained almost exclusively green,

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.3

indicating an uptake of more Syto-9 than PI (Figure 1b, IV-VI). The sporulation process is completed when the mothercell undergoes autolysis to release the mature spore. Maturefree spores could be seen as early as hour 44 (Figure 1b, V).Later, around hour 58 (Figure 1b, VI), a portion of the cellsbecame motile again. Though these cells appear like vegeta-tive cells, they stained predominantly green, instead of red,and did not produce appreciable amounts of acid. We hypoth-esize that this staining change reflects modifications in mem-brane composition due to different environmental conditions(presence of solvents and other metabolites) rather than cellviability and assume that this newly identified cell type hasdifferent transcriptional characteristics, which we testednext.

The transcriptional program of clostridial differentiationTo ensure that important transcriptional, physiological, andmorphological changes were captured [7,8], RNA sampleswere taken every hour during exponential phase and everytwo hours after that until late stationary phase when sam-pling frequency decreased. mRNA from 25 timepoints (Fig-ure 1a) were selected for transcriptional analysis byhybridizing pairs of 22k oligonucleotide microarrays on a dyeswap configuration using an mRNA pool as reference. Therewere 814 genes, or 21% of the genome, that surpassed thethreshold of expression in at least 20 of the 25 microarray

Morphological and gene expression changes C. acetobutylicum undergoes during exponential, transitional, and stationary phasesFigure 1Morphological and gene expression changes C. acetobutylicum undergoes during exponential, transitional, and stationary phases. (a) Growth and acid and solvent production curves as they relate to morphological and transcriptional changes during sporulation. The gray bar indicates the beginning of the transitional phase as determined by solvent production. A600 with microarray sample (filled squares); A600 (open squares); butyrate (filled circles); butanol (filled triangles). Roman numerals correspond with those in (b), and bars and numbers along the top correspond to the clusters in (c). (b) Morphological changes during sporulation. When stained with Syto-9 (green) and PI (red), vegetative cells take on a predominantly red color (I and II). At peak butanol production, swollen, cigar-shaped clostridial-form cells appear (arrow in III), which stain almost equally with both dyes, and persist until late stationary phase. Towards the end of solvent production (IV), endospore (arrow 1) forms are visible, and clostridial (arrow 2) forms are still present. As the culture enters late stationary phase (V and VI), cells stain almost exclusively green, regardless of morphology. All cell types are still present, including free spores (arrows in V and VI), and vegetative cells identified by their motility. (c) Average expression profiles for each K-means cluster generated using a moving average trendline with period 3. (d) Expression of the 814 genes (rows) at 25 timepoints (columns, hours 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 48, 54, 58, and 66). Genes with higher expression than the reference RNA are shown in red and those with lower expression as green. Saturated expression levels: ten-fold difference.

Exponential (1)Vegetative form

134 genes (hour 6-10)

Transitional (2)Vegetative form

139 genes (hour 10-18)

Stationary (3)Clostridial form

175 genes (hour 18-36)

Early stationary (4)Clostridial form

84 genes (hour 18-24)

Middle stationary (5)Clostridial form

120 genes (hour 24-36)

Late stationary (6)Endospore/free spore

162 genes (hour 36-66)

6 12 22 66

Time (h)

I

VV I

II

IV

III

1

2

10

1.0

0.1

0.01

100

A6

00

10 20 30 40 50 60

Time (h)

0

50

100

150

200

Con

cent

ratio

n (m

M)

1 2 4 5 6

3 (c) (d)(a)

I II III IV

V VI

(b)

32 446 12 22 66

Time (h)32 44

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.4

timepoints and had two or more timepoints differentiallyexpressed at a 95% confidence level [11]; these genes wereclassified as having a temporal differential expression profile.We chose these strict selection criteria in order to robustlyidentify the key expression patterns of the differentiationprocess. We relaxed these criteria in subsequent gene ontol-ogy-driven analyses. Expression data were extensively vali-dated by, first, quantitative reverse transcription PCR (Q-RT-PCR) analysis (focusing on key sporulation factors) from a

biological replicate culture (Figure 2), and, second, by sys-tematic comparison to our published (but limited in scopeand duration) microarray study (see Additional data file 1 forFigure S1 and discussion).

Six distinct clusters of temporal expression patterns wereselected (Figure 1c,d) by K-means to achieve a balancebetween inter- and intra-cluster variability. To examine tran-

Q-RT-PCR and microarray data comparisonFigure 2Q-RT-PCR and microarray data comparison. RNA from a biological replicate bioreactor experiment was reverse transcribed into cDNA for the Q-RT-PCR. All expression ratios are shown relative to the first timepoint for both Q-RT-PCR (open circles) and microarray data (filled squares). Asterisks represent data below the cutoff value for microarray analysis. Samples were taken every six hours starting from hour 6 and continuing until hour 48. The genes examined were from several operons with different patterns of expression.

* * * **

*

* * ** * * * *

Exp

ress

ion

ratio

rel

ativ

e to

firs

t tim

epoi

nt

abrBsinR

spo0A sigE

sigG

sigF

spoIIE

sigK

spoIIIDspoIIIAA

100

10

1

0.1100

10

1

0.110

1

0.1

100

10

1

0.1

1,000

24 36 48

Time (h)

0.01

120

24 36 48120 24 36 48120

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.5

scriptional changes in larger functional groups (for example,transcription, motility, translation), each cluster was ana-lyzed according to the Cluster of Orthologous Groups ofproteins (COG) classification [12] and the functional genomeannotation [13]. To determine if a COG functional group wasoverrepresented in any of the K-means clusters, first the per-centage of each group in the genome was determined, andthen the percentage of each group was determined in each ofthe K-means clusters. By comparing the percentage in the K-means clusters to the genome percentage, we could identifyoverrepresented groups (Additional data file 2).

Exponential phase: motility, chemotaxis, nucleotide and primary metabolismThe first cluster contains 134 genes highly expressed duringexponential growth (hours 6 to 10; see Additional data file 2for a list of the genes). This cluster characterizes highly motilevegetative cells (Figure 1b, I) and, given the minimal amountof knowledge on the genes responsible for motility and chem-otaxis in clostridia, our analysis offers the possibility of iden-tifying these genes at the genome scale [14]. This clusterincludes the flagella structural components flagellin and flbD,the main chemotaxis response regulator, cheY (CAC0122;responsible for flagellar rotation in B. subtilis [15]), as well asseveral methyl-accepting chemotaxis receptor genes(CAC0432, CAC0443, CAC0542, CAC1600, CAP0048). COGanalysis showed that genes related to cell motility (COG classN) and nucleotide transport and metabolism (COG class F)were overrepresented in this cluster (Additional data file 2).In order to investigate cell motility further, all genes that fellwithin this COG class were hierarchically clustered accordingto their expression profiles (see Additional data file 3 for Fig-ure S2 and discussion). Interestingly, the two main cell motil-ity gene clusters, the first including most of the flagellarassembly and motor proteins and the second containing mostof the known chemotaxis proteins, clustered together and dis-played a bimodal expression pattern (Figure S2). The geneswere not only expressed during exponential phase but alsoduring late stationary phase, around hour 38, which is con-sistent with the observation that a motile cell population wasagain observed in late stationary phase. Included in the cate-gory of nucleotide transport and metabolism are severalpurine and pyrimidine biosynthesis genes: a set of five con-secutive genes, purECFMN, the bi-functional purQ/L gene,purA, pyrPR, pyrD, and pyrI. Two other purine synthesisgenes (purH, purD) showed very similar profiles but were notclassified within this cluster by the clustering algorithm. Veg-etative cells, which correspond to this cluster, produce ATPthrough acidogenesis, whereby the cells uptake glucose andconvert it to acetic and butyric acid. Because glucose is themain energy source, multiple genes for glucose transportwere included within this cluster, including the glucose-spe-cific phosphotransferase gene, ptsG, the glucose kinase glcKand CAP0131, the gene most similar to B. subtilis glucose per-mease glcP. The genes required for the metabolism of glucoseto pyruvate did not show temporal regulation, suggesting that

expression of these genes is constitutive-like (see Additionaldata file 3 for Figure S3 and discussion). Acetic acid produc-tion genes pta and ack were not temporally expressed, butbutyrate production genes ptb and buk were. Thoughexpressed throughout exponential phase, the expression ofboth ptb and buk slightly peaked during late exponentialphase, as previously seen [7], and thus fall in the transitional(second) cluster. Analysis of the expression patterns of all thegenes involved in acidogenesis, not just the differentiallyexpressed genes discussed here, is included in Figure S3 inAdditional data file 3. Finally, the expression patterns of thetwo classes of hydrogenases (iron only and nickel-iron) wereinvestigated (Figure S3 in Additional data file 3). hydA, theiron only hydrogenase that catalyzes the production of molec-ular hydrogen, was expressed only during exponential phase,whereas the iron-nickel hydrogenase, mbhS and mbhL, wasexpressed throughout stationary phase.

Initiation of sporulation: abrB, sinR, lipid and iron metabolismThe transitional phase is captured by 139 genes in the secondcluster (Figure 1c,d; Additional data file 2). It is made up ofgenes that show elevated expression between hours 10 and 18and is when solvent formation was initiated. This clustercharacterizes the shift from vegetative cells to cells commit-ting to sporulation and thus includes two important regula-tors of sporulation, abrB (CAC0310) and sinR (CAC0549),which are discussed in more detail below. Also characteristicof this shift from vegetative growth to sporulation was theoverrepresentation of genes related to energy production andconversion (COG class C), since sporulation is an energyintensive process. Solvent production began in the transi-tional phase, though the genes responsible for solvent pro-duction fall in the next (third) cluster; the third clusterpartially overlaps with this second cluster but is distinguishedby a sustained expression pattern. In response to these sol-vents, C. acetobutylicum undergoes a change in its mem-brane composition and fluidity, generally decreasing the ratiobetween unsaturated to saturated fatty acids [16-18]. Consist-ent with this change, genes related to lipid metabolism (COGclass I) were overrepresented in this cluster. To further inves-tigate this COG class, all genes identified as COG class I werehierarchically clustered (see Additional data file 3 for FigureS4 and discussion). Seven genes that were upregulated justbefore the onset of sporulation fall within the same operonand are related to fatty acid synthesis. In contrast, many ofthe most characterized genes involved in fatty acid synthesis(accBC, fabDFZ, and acp) maintain a fairly flat profilethroughout the timecourse (Figure S4 in Additional data file3). Also within this cluster is the gene responsible for cyclo-propane fatty acid synthesis (cfa), though classified in COGclass M (cell envelope biogenesis) and not COG class I.Importantly, the ratio of cyclopropane fatty acids in the outermembrane has been shown to increase as cells enter station-ary phase [18,19], but the overexpression of this gene alonewas unable to produce a solvent tolerant strain [19]. Thoughnot overrepresented in this cluster, all the genes within COG

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.6

class M were also hierarchically clustered (see Additional datafile 3 for Figure S5 and discussion). The transitional clusteralso included several genes related to iron transport andregulation like the fur family iron uptake regulator CAC2634,the iron permease CAC0788, feoA, feoB, fhuC, and two iron-regulated transporters (CAC3288, CAC3290), which is con-sistent with the earlier, more limited data [7]. Significantly,iron-limitation has been found to promote solventogenesis[20].

Solventogenesis, clostridial form, stress proteins, and early sigma factorsThe third cluster (Figure 1c,d; Additional data file 2) of 175upregulated genes represents the solventogenic/stationaryphase as it contains all key solventogenic genes. This clustercharacterizes the transcriptional pattern of clostridial cells,the unique developmental stage in clostridia and firstrecognizable cell type of the sporulation cascade, and exhib-ited a longer upregulation of gene expression than the previ-ous two clusters. Indeed, its range overlapped the previous(second) and the next two (fourth and fifth) clusters. Theclostridial form is generally recognized to be the form respon-sible for solvent production [8,21] and is distinguished mor-phologically as swollen cell forms with phase bright granulosewithin the cell [21]. This cluster captures both of these char-acteristics with the inclusion of the solventogenic genes andseveral granulose formation genes. The solventogenic genesadhE1-ctfA-ctfB, adc, and bdhB were initially induced duringtransitional phase, the second cluster, but were expressedthroughout stationary phase and were thus placed within thiscluster. Two granulose formation genes, glgC (CAC2237) andCAC2240, and a granulose degradation gene, glgP(CAC1664), were included within this cluster. The other twogranulose formation genes, glgD (CAC2238) and glgA(CAC2239), though not included in this cluster, displayed asimilar expression profile to glgC and CAC2240. The con-comitant requirement of NADH during butanol productiondrove the expression of three genes involved in NAD forma-tion: nadABC. Expression of the stress-response gene hsp18,a heat-shock related chaperone, and the ctsR-yacH-yacI-clpC operon, containing the molecular chaperone clpC andthe stress-gene repressor ctsR, also fell in this cluster and par-alleled the expression of the solventogenic genes (see Addi-tional data file 3 for Figure S6). Other important stress-response genes, groEL-groES (CAC2703-04) and hrcA-grpE-dnaK-dnaJ (CAC1280-83), mirrored this expressionpattern, though were not differentially expressed according tothe strict criteria employed for selecting the genes of Figure2c,d (Figure S6 in Additional data file 3). Although genesencoded on the pSOL1 megaplasmid [22] represent less than5% of the genome, they constitute 15% of genes in this cluster.pSOL1 harbors all essential solvent-formation genes and,importantly, some unknown gene(s) essential for sporulation[22]. Besides the genes listed in this cluster, the vast majorityof the genes located on pSOL1 were expressed throughout sta-tionary phase, with most being upregulated at the onset of

solventogenesis (see Additional data file 3 for Figure S7). Sev-eral key sporulation-specific sigma factors (σF, σE, σG) and theσF-associated anti-sigma factors in the form of the tricistronicspoIIA operon (CAC2308-06) belong to this cluster alongwith one of the two paralogs of spoVS (CAC1750) and one ofthree spoVD paralogs (CAP0150). The second spoVS paralog(CAC1817) did not meet the threshold of expression in 12 ofthe 25 timepoints; the other two paralogs of spoVD(CAC0329, CAC2130) were above the expression cutoff butdid not show significant temporal regulation. Of unknownsignificance was the expression of a large cluster of genesinvolved in the biosynthesis of the branched-chain aminoacids valine, leucine and isoleucine (CAC3169-74) coincidingwith the onset of solventogenesis, as shown before [7,23], aswell as the upregulation of several glycosyltranferases (seeAdditional data file 3 for Figure S8). The upregulation ofvaline, leucine, and isoleucine synthesis genes could be indic-ative of a membrane fluidity adaptation [7]. In B. subtilis,these branched-chain amino acids can be converted intobranched-chain fatty acids and change the membrane fluidity[24], and under cold shock stress, B. subtilis downregulates anumber of genes related to valine, leucine, and isoleucine syn-thesis [25]. Therefore, this upregulation may be anothermechanism to change membrane fluidity, though the ratio ofunbranched and branched fatty acids has not been reportedin studies investigating membrane composition [16-18,26].

Stationary phase carbohydrate (beyond glucose) and amino acid metabolismThe fourth cluster (Figure 1c,d; Additional data file 2) of 84genes represents a sharp induction of expression between 18and 24 hours (early stationary phase). This cluster falls withinthe stationary (third) cluster described above. This is a com-pact group, with 70% belonging to one of three COG catego-ries: carbohydrate transport and metabolism, transport andmetabolism of amino acids, and inorganic ion transport andmetabolism. A number of different carbohydrate substratepathways, from monosaccharides (fructose, galactose, man-nose, and xylose) to disaccharides (lactose, maltose, andsucrose) to complex carbohydrates (cellulose, glycogen,starch, and xylan), were investigated, and many exhibitedupregulation during stationary phase, though only a few arehighly expressed (see Additional data file 3 for Figure S9).The significance of this upregulation of non-glucose pathwaysis unknown, because sufficient glucose remains in the media(approximately 200 mM or about 44% of the initial glucoselevel). Of particular interest was the upregulation of severalgenes related to starch and xylan degradation (Figure S9 inAdditional data file 3). The two annotated α-amylases(CAP0098 and CAP0168) along with the less characterizedglucosidases and glucoamylase were all upregulated through-out stationary phase and a number were highly expressed,like CAC2810 and CAP0098. Also upregulated were the pre-dicted xylanases CAC2383, CAP0054, and CAC1037, withCAP0054 and CAC1037 being highly expressed during sta-tionary phase. Mirroring this pattern were CAC1086, a xylose

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.7

associated transcriptional regulator, and the highly expressedCAC2612, a xylulose kinase. The genes related to glycogenmetabolism are believed to be involved in granuloseformation, as discussed earlier. Several genes for argininebiosynthesis (argF, argGH, argDB, argCJ, carB) wereinduced during this time, probably as a result of its depletionin the culture medium.

Genes underlying the activation of the sporulation machinery and the genes for tryptophan and histidine biosynthesisThe fifth cluster (Figure 1c,d; Additional data file 2), repre-senting the middle stationary phase, contains 120 genesmainly expressed between hours 24 and 36, and again fallswithin the stationary (third) cluster described above. Most ofthe genes in this cluster activate the sporulation-relatedsigma factors (σF, σE, σG) or are putatively regulated by them.These include spoIIE, the phosphatase that dephosphorylatesSpoIIAA and results in the activation of σF, and the σE-dependent operons spoVR (involved in cortex synthesis),spoIIIAA-AH (required for the activation of σG), and spoIVA(involved in cortex formation and spore coat assembly). TheσG-dependent spoVT gene has two paralogs in C. acetobutyl-icum (CAC3214, CAC3649); the transcriptional pattern sug-gests that CAC3214, included in this cluster, is the real spoVT.Sporulation-related genes included in this cluster are threecotF genes, one cotJ gene, one cotS gene, the spore matura-tion protein B, a small acid soluble protein (CAC2365), andtwo spore lytic enzymes (CAC0686, CAC3244). Though sev-eral sporulation-related genes are included in the next (sixth)cluster as well, most, beyond those listed here, are upregu-lated in mid-stationary phase (see Additional data file 3 forFigure S10 and discussion). Seven genes of the putativeoperon (CAC3157-63) encoding genes for tryptophan synthe-sis from chorismate and ten genes for histidine synthesis(CAC0935-43, CAC3031) were also included here.

Spore maturation and late-stationary phase vegetative cellsThe sixth cluster, representative of the late stationary phase,includes 162 genes mainly expressed after hour 36 (Figure1c,d; Additional data file 2). This cluster captured the expres-sion profiles of the forespore and endospore forms, freespores, and late-stage vegetative-like cells. The endosporeform represents the last stage before mature spores arereleased, and therefore fewer sporulation-related genes arewithin this cluster than previous ones. The sporulation-related genes included in this cluster are two small acid-solu-ble proteins (CAC1522 and CAC2372), a spore germinationprotein (CAC3302), a spore coat biosynthesis protein(CAC2190) and a spore protease (CAC1275). Also within thiscluster are the two phosphotransferase genes, CAC2958 (agalactitol-specific transporter) and CAC2965 (a lactose-spe-cific transporter), another annotated cheY (CAC2218), vari-ous enzymes related to different sugar pathways (CAC2180,CAC2250, CAC2954), and two glycosyltransferases(CAC2172, CAC3049). Expression of these genes may bereflective of the late-stage vegetative-like cells observed dur-

ing microscopy and demonstrate they have a different geneticprofile compared to the early vegetative cells. Interestingly,this cluster is enriched in defense mechanism genes (COGclass V) like a phospholipase (CAC3026) and multidrugtransporters that may play a role in resistance to a variety ofenvironmental toxins.

General processes: cell division and ribosomal proteinsTwo additional gene classes (cell division and ribosomal pro-teins), though not overrepresented in any of the six clustersdescribed above, were investigated because of their impor-tance in cellular processes and interesting expression pat-terns. COG class D (cell division and chromosomepartitioning), besides important genes for vegetative sym-metric division, includes ftsAZ, important for both symmetricand asymmetric cell division, and soj (a regulator of spo0J)and spoIIIE, important for proper chromosomal partitioningbetween the mother cell and prespore. These genes, alongwith several uncharacterized genes, were upregulated at thebeginning of sporulation (see Additional data file 3 for FigureS11). Almost all the ribosomal proteins were downregulatedas the culture entered stationary phase, and interestingly,about half of those downregulated genes were again upregu-lated in mid-stationary phase and remained upregulated untillate-stationary phase (see Additional data file 3 for FigureS12). This upregulation is likely related to the late-stage veg-etative-like cells seen.

Expression and activity patterns of sporulation-related sigma factors and related genesExpression of sporulation transcription factorsSporulation in bacilli is initiated by a multi-component phos-phorelay [27], which is absent in clostridia, but the masterregulator of sporulation, Spo0A, is conserved [1,13]. Briefly,in B. subtilis, phosphorylated Spo0A promotes the expressionof prespore-specific sigma factor σF and mother cell-specificsigma factor σE [28]. σF is followed by σG, which is controlledby both σF and σE, and σE is followed by σK, which is control-led by σE and SpoIIID [28]. sigH expression, in bacilli, isinduced before the onset of sporulation and aids spo0A tran-scription [28]. Here, sigH expression underwent a modesttwo-fold induction, relative to the first timepoint, during theonset of sporulation but never increased beyond three-fold, incontrast to all other sporulation factors (Figure 3a). spo0Aexpression also peaked during the onset of sporulation at over12-fold and maintained a minimum of 3-fold induction untilhour 36 (Figure 3a,b). Once phosphorylated, in bacilli andlikely in C. acetobutylicum [29], Spo0A regulates the expres-sion of the operons encoding sigF, sigE, and spoIIE [30], thelatter of which acts as an activator of σF. sigF and sigE exhib-ited an initial 16- and 8-fold induction, respectively, at hour12, the timing of peak spo0A expression, but a second higherlevel of induction, 46- and 66-fold, respectively, was reachedlater at hour 24 (Figure 3c) and confirmed with Q-RT-PCR(Figure 2). The plateau or decrease in expression of spo0A,sigF, and sigE coincided with the peak expression of two

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.8

known repressors, abrB and sinR, of sporulation genes in B.subtilis (Figure 3b), the former repressing the expression ofspo0A promoters and the latter directly binding to thepromoter sequences of the spo0A, sigF, and sigE operons[31,32]. C. acetobutylicum contains three paralogs of abrB,among which CAC0310 exhibited the highest promoter activ-ity and, when downregulated, causes delayed sporulation anddecreased solvent formation [33]. sinR (CAC0549) expres-sion in C. acetobutylicum was previously reported [33] to beweak, but our data show a significant amount of expressionand suggest a similar role as that in B. subtilis. In B. subtilis,Spo0A either indirectly (sinR) or directly (abrB) repressesthe genes of these two repressors [32,34]. The expression pat-terns of both genes did decrease after peak Spo0A~P deducedactivity (Figure 4b; see below), indicating a similar regulatorynetwork may be involved in C. acetobutylicum. sigF, sigE andsigG have very similar expression patterns (Figure 3c). BothsigF and sigE are activated by Spo0A~P, so similar expres-sion profiles were expected. In B. subtilis, a sigG transcript isalso detected early, but this transcript is read-through fromsigE, located immediately upstream of sigG, and is not trans-lated [35,36]. Translation of sigG occurs when the gene isexpressed as a single cistron from a σF-dependent promoterlocated between sigE and sigG [35,36]. In C. acetobutylicum,sigE and sigG are also located adjacent to each other, but a σF

promoter was not predicted between the two genes [37].Thus, it was predicted that sigG is only expressed as part ofthe sigE operon (consisting of spoIIGA, the processingenzyme for σE, and sigE). Our transcriptional data seem tosupport this prediction because all three genes, spoIIGA,sigE, and sigG, have very similar transcriptional patterns(Figure 3f), suggesting they are expressed as a single tran-script, like the spoIIAA-spoIIAB-sigF operon (Figure 3e).However, from Northern blots probing against sigE-sigG,three separate transcripts were seen: one for spoIIGA-sigE-sigG, one for spoIIGA-sigE, and one for sigG [29]. Unfortu-nately, the current data cannot resolve this issue definitively,since the microarrays only detect if a transcript is present ornot.

Deduced activity profiles of sporulation factorsWe also desired to estimate the activity profiles for the keysporulation factors (σH, Spo0A, σF, σE, and σG; Figure 4). Wedid so by averaging the expression profiles of known orrobustly identifiable canonical genes of their regulons [1]. Toadjust for differences in relative expression levels, expressionprofiles were standardized before averaging [7]. This is a sur-rogate reporter assay, which we believe is as accurate as mostreporter assays. For a detailed discussion of the genes used toconstruct the plots, see Additional data file 4. For all of theplots (Figure 4), peak activity took place after peak expres-sion, as expected. Of all the factors, σH activity peaked first,during early transitional phase, and this was followed by adecrease in activity until stationary phase, when activityincreased again (Figure 4a,f). Spo0A~P activity was the nextto peak, during late transitional phase, and stayed fairly con-

Investigation of the sporulation cascade in C. acetobutylicumFigure 3Investigation of the sporulation cascade in C. acetobutylicum. (a-f) Expression profiles of sporulation genes shown as ratios against the first expressed timepoint. (a) The first three sporulation factors: spo0A (red filled triangles), sigH (black filled squares), and sigF (open blue circles). (b) spo0A (red filled triangles) and possible sporulation regulators: abrB (open black circles) and sinR (green filled diamonds). (c) Sporulation factors downstream of spo0A: sigF (open blue circles), sigE (black filled triangles), and sigG (open red squares). (d) Genes related to sigK expression: spoIIID (blue filled diamonds), yabG (red filled triangles), and spsF (black filled triangles). (e) spoIIA operon: spoIIAA (black filled diamonds), spoIIAB (red filled triangles), and sigF (open blue circles). (f) spoIIG operon and sigG: spoIIGA (green filled diamonds), sigE (black filled triangles), and sigG (open red squares). The gray bar indicates the onset of transitional phase. (g) Ranked expression intensities. White denotes a rank of 1, while dark blue denotes a rank of 100 (see scale). Gray squares indicate timepoints at which the intensity did not exceed the threshold value. Bracketed genes are predicted to be coexpressed as an operon.

(a) (b)

(c)

Exp

ress

ion

ratio

100

10

1

0.1

100

10

1

0.1

100

10

1

0.1

1,000

0 12 24 36 48 60 0 12 24 36 48 60

0 12 24 36 48 60

(g) Time

6 10 18 26 34 44 66

1 50 100Rank scale

0 12 24 36 48 60

100

10

1

0.1

(d)

CAC2071 - spo0ACAC0310 - abrBCAC0549 - sinRCAC3152 - sigHCAC2308 - spoIIAACAC2307 - spoIIABCAC2306 - sigFCAC1694 - spoIIGACAC1695 - sigECAC1696 - sigGCAC3205 - spoIIECAC2898 - spoIIRCAC2093 - spoIIIAACAC2092 - spoIIIABCAC2091 - spoIIIACCAC2090 - spoIIIADCAC2088 - spoIIIAFCAC2087 - spoIIIAGCAC2086 - spoIIIAHCAC2859 - spoIIIDCAC2905 - yabGCAC2190 - spsF

(e)

0 12 24 36 48 60

10

1

0.1

100

0 12 24 36 48 60

10

1

0.1

100

1,000 (f)

Time (h) Time (h)

Time (h)

Exp

ress

ion

ratio

Exp

ress

ion

ratio

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.9

stant throughout the rest of the timecourse (Figure 4b,f). σF

activity had an initial induction during transitional phase, butthen stayed constant until 24 hours (Figure 4c,f). After 24hours, the activity increased again and stayed fairly constantat this higher activity level for the rest of the culture. σE activ-ity increased slightly during late transitional phase, but itsmajor increase occurred after 24 hours during mid-stationaryphase (Figure 4d,f). Like the previous sigma factors, σG activ-ity increased throughout early stationary phase and earlymid-stationary phase, but the major increase occurred afterhour 30 (Figure 4e,f). The activity of all of the factors, exceptfor Spo0A and σF, decreased during late stationary phase athour 38. σG activity began to increase slightly again at hour 48but did not peak again. Considering only major peaks in activ-ity, the Bacillus model of sporulation is generally true with

the peaks progressing from σH to Spo0A~P to σF to σE andfinally to σG (Figure 4f).

Can we deduce the activation and processing of σF, σE, and σG from transcriptional data?In B. subtilis, the sigma factors downstream of Spo0A (σF, σE,and σG) are all regulated by a complex network of interactions[1]. We desired to examine if our transcriptional data could beused to do a first test to determine whether the mechanismsemployed in the B. subtilis model are valid for C. acetobutyl-icum. In B. subtilis, σF is held inactive in the pre-divisionalcell by the anti-σF factor SpoIIAB. σF is released when theanti-anti-σF factor SpoIIAA is dephosphorylated by SpoIIE,resulting in SpoIIAA binding to SpoIIAB, which then releases

Transcriptional and putative activity profiles for the major sporulation factorsFigure 4Transcriptional and putative activity profiles for the major sporulation factors. The standardized expression ratios compared to the RNA reference pool of (a) sigH, (b) spo0A, (c) sigF, (d) sigE, and (e) sigG are shown in black, while the activity profiles based on the averaged standardized profiles of canonical genes under their control are shown in red. Putative genes (based on the B. subtilis model) responsible for activating σF (spoIIE), σE (spoIIR), and σG (spoIIIA operon) are shown as light blue diamonds. For the spoIIIA operon, the individual standardized ratios (Figure S13g in Additional data file 4) were averaged together. The gray bar indicates the onset of the transitional phase. (f) Compilation of the activity profiles for sigH (red), spo0A (blue), sigF (green), sigE (black), and sigG (purple). The numbers along the top correspond to the clusters in Figure 1c,d and the bars indicate the timing of each cluster.

(a) (b) (c)

(d)

(e)

Exp

ress

ion

ratio

1.6

1.3

1.0

0.8

0 12 24 36 48 60

0 12 24 36 48 600 12 24 36 48 60 0 12 24 36 48 60

0 12 24 36 48 60

Time (h)

0 12 24 36 48 60

0.6

1.6

1.3

1.0

0.8

0.6

1.6

1.3

1.0

0.8

0.6

1.6

1.3

1.0

0.8

0.6

1.6

1.3

1.0

0.8

0.6

(f)1.6

1.3

1.0

0.8

0.6

1 2 3

4 5

6

2.1

Exp

ress

ion

ratio

Exp

ress

ion

ratio

Time (h) Time (h)

Time (h)

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.10

σF. In C. acetobutylicum, spoIIAB (CAC2307) and spoIIAA(CAC2308) are transcribed on the same operon as sigF (Fig-ure 3e), but spoIIE (CAC3205) is transcribed separately. Theinitial increase in σF activity during the transitional phase wasnot accompanied by an increase in spoIIE expression, but thepeak in σF activity did occur after spoIIE upregulation (Figure4c). Despite the sustained level of σF activity, sigF and spoIIEdecreased in expression, though spoIIE expression didincrease slightly again after 48 hours (Figure 4c). In B. subti-lis, the pro-σE translated from the sigE gene undergoesprocessing from SpoIIGA, which must interact with SpoIIR inorder to accomplish the σE activation. In C. acetobutylicum,SpoIIGA (CAC1694) is transcribed on the same operon assigE (Figure 3f), and SpoIIR is coded by CAC2898. σE activityincreased with the induction of spoIIR (Figure 4d), suggest-ing a similar mechanism as in B. subtilis. Finally, σG activa-tion in B. subtilis is dependent upon the eight genes withinthe spoIIIA operon. Here, the second and larger increase inσG activity followed peak expression of the spoIIIA operon,but the early increase in σG activity was not characterized by alarge induction of spoIIIA expression (Figure 4e). We tenta-tively conclude that the B. subtilis processing and activationmodel does generally hold true in C. acetobutylicum, but fur-ther investigation is needed to determine the exact timing andinteraction of the various factors and their activators.

Is there a functional sigK?In B. subtilis, σK is formed by splicing together two genes(spoIVCB and spoIIIC), both under the control of σE andSpoIIID [38], separated by a skin element [39]. In contrast, asingle gene encoding σK has been annotated in C.acetobutylicum [13]. The gene was initially identified using aPCR-approach [40] and was later detected by primer exten-sion in a phosphate-limited, continuous culture of C. aceto-butylicum DSM 1731 [41]. spoIIID, which controls sigKexpression with σE in B. subtilis, reached peak expression athour 30, which is consistent with it being under σE control(Figure 3d) [42]. However, at no timepoint in this study didsigK exceed the cutoff expression criterion. Q-RT-PCR alsoshowed a significantly lower sigK induction compared to theother sigma factors and suggests the transcript, if expressed,is at much lower levels than any other gene analyzed (Figure2). The putative main σK processing enzyme, SpoIVFB(CAC1253), also did not exceed the cutoff criterion. To helpdetermine if there is an active σK, we investigated two genescontrolled by σK in B. subtilis. yabG (CAC2905), whichencodes a protein involved in spore coat assembly, was upreg-ulated mid-stationary phase and peaked at hour 30 (Figure3d), and spsF (CAC2190), involved in spore coat synthesis,was not upregulated until late stationary phase, at hour 38(Figure 3d). From these two genes, it is difficult to determinewhether a functional sigK gene exists or not. Clearly they areboth transcribed, but based on its expression pattern, yabGcould fall under the control of σE instead of σK. spsF upregu-lation is late enough to possibly indicate σK regulation though.Ideally, more genes need to be investigated to draw firmer

conclusions, but because few σK regulon homologs exist in C.acetobutylicum, we cannot currently determine if there is σK

activity or not.

Distinct profiles of sensory histidine kinases: which for Spo0A?Revisiting the orphan kinasesAs discussed, phosphorylated Spo0A is responsible for initi-ating sporulation in both bacilli and clostridia along with sol-vent formation in C. acetobutylicum. In bacilli, Spo0A isphosphorylated via a multi-component phosphorelay [43],initiated by five orphan histidine kinases, KinA-E (kinasesthat lack an adjacent response regulator); this phosphorelaysystem is absent in all sequenced clostridia [1]. Alternatively,Spo0A in clostridia may be directly phosphorylated by a his-tidine kinase, orphan or not, as was hypothesized in [1,7].This alternative was demonstrated in C. botulinum, where theorphan kinase CBO1120 was able to phosphorylate Spo0A[44]. In C. acetobutylicum, five true orphan kinases havebeen identified with a sixth orphan, CAC2220, identified asCheA, which has a known response regulator [1].

A kinase that could directly phosphorylate Spo0A is expectedto have a peak in expression before or during the activation ofSpo0A, as the orphan kinases in B. subtilis do [45-47]. As ameasure of Spo0A activity, the expression of the sol operon(CAP0162-64) was used, as before [7], because it is inducedby Spo0A~P. The initial induction of the sol operon, almost100-fold, occured at hour 10 (before spo0A reached it maxi-mum expression), with detectable levels of butanol appearingbefore the second induction of the sol operon. This secondinduction, of another 10-fold, followed the peak in spo0Aexpression (Figure 5a). It is clear that some level of phospho-rylated Spo0A exists at 10 hours; therefore, kinase candidatesmust display an increase in expression before 10 hours. Of thefive orphan kinases (Figure 5b,c), CAC2730 displayed the ear-liest peak followed by CAC0437, CAC0903, and CAC3319.CAC0323 never displayed a prominent peak in expressioneither before or after sol operon induction (Figure 5b) andlikely does not play a role in phosphorylating Spo0A. Of theremaining four, CAC0437 and CAC2730 peaked only oncebefore the initial sol operon induction, while CAC0903peaked before each induction of the sol operon (Figure 5b,c).CAC3319 expression slightly mirrored that of the sol operon,with an increase before initial induction followed by a pla-teau, and an increase in expression again until it peaked justafter the sol operon peaked (Figure 5c). The proteins encodedby CAC0437 and CA0903 displayed the most similarity to theprotein encoded by CBO1120, the orphan kinase in C. botuli-num shown to phosphorylate Spo0A [44].

Non-orphan kinase expressionThough primarily interested in orphan kinases because of thesimilarity to the B. subtilis model, a two-component responsesystem could also be responsible for the phosphorylation ofSpo0A. The remaining 30 annotated histidine kinases were

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.11

also investigated to determine if any displayed a peak inexpression before the initial induction of the sol operon(Additional data file 5). Six kinases (Figure 5d,e) were foundto have a peak in expression at 8 hours. CAC0290 andCAC3430 subsequently decreased in expression whileCAC0225 and CAC0863 maintained expression at initial lev-els. Despite a dip in expression at hour 9, CAC1582 main-tained an increased expression level from 8 hours on.CAC2434 peaked at hour 8, dropped back to initial levels, butthen steadily increased with the second induction of the soloperon.

Sigma factors of unknown function: a first assessment of their functional rolesSeventeen sigma factors are annotated on the C. acetobutyli-cum genome, including two on pSOL1. Two, sigK (CAC1689)and CAC1770 (a sigK-like sigma factor), are expressed at very

low levels and two others, CAC1509 (annotated 'specializedsigma subunit of RNA polymerase') and CAC1226 (one of twoannotated sigAs), are only above the expression cutoff in 8out of 25 timepoints, and these timepoints are notconsecutively expressed. Among the expressed sigma factors,six, CAP0157, CAP0167, CAC3267, CAC1766, CAC2052, andCAC0550, are of unknown function, while the remainingseven expressed sigma factors (σH, σF, σE, σG, σA, σD, and σ54/rpoN) are of predicted known function. To assess the poten-tial role of the remaining six sigma factors of unknown func-tion, we examined the transcriptional profiles (Figure 6a,b)and probed the binding motifs in their promoter regions forpredicted Spo0A, σA, σE, and σF/σG binding motifs [37].

Transcriptional analysis of the sigma factors of unknown functionLoss of pSOL1 impairs sporulation at the level of spo0Aexpression [7,48], thus generating increased interest for

Expression profiles of uncharacterized sensory histidine kinases that could phosphorylate Spo0AFigure 5Expression profiles of uncharacterized sensory histidine kinases that could phosphorylate Spo0A. Gene and operon profiles are ratios compared against the first expressed timepoint. Gray bar indicates the onset of the transitional phase. (a) Activation of Spo0A as represented through the upregulation of the sol operon (black filled diamonds; CAP0162-164) and the production of butanol (green crosses). Activation occurs before spo0A (red filled triangles) reaches peak expression. (b) Expression of the orphan kinases CAC0323 (blue filled diamonds), CAC0437 (green filled triangles), and CAC0903 (red filled circles) relative to the sol operon (black filled diamonds) (right-hand side vertical axis). (c) Expression of the orphan kinases CAC2730 (blue filled squares) and CAC3319 (open red circles) relative to the sol operon (black filled diamonds) (right-hand side vertical axis). (d) Expression of the two-component kinases CAC0225 (green filled circles), CAC0290 (red filled squares), and CAC0863 (open blue diamonds) relative to the sol operon (black filled diamonds) (right-hand side vertical axis). (e) Expression of the two-component kinases CAC1582 (green filled squares), CAC2434 (open blue circles), and CAC3430 (open red diamonds) relative to the sol operon (black filled diamonds) (right-hand side vertical axis). (f) Ranked expression intensities. White denotes a rank of 1, while dark blue denotes a rank of 100 (see scale). Plot covers the entire timecourse, whereas the previous figures only covered the first 14 hours. Gray squares indicate timepoints at which the intensity did not exceed the threshold value.

(a) (b) (c)

(d)

100

10

1

0.1

1,000

5 10 15 20

20

0

40

60

80

Exp

ress

ion

ratio

Concentration (m

M)

100

10

1

0.1

1,000

100

10

1

0.1

1,000

100

10

1

0.1

1,000

5 10 15 20

Time (h)

5 10 15 20 5 10 15 20

10

1

0.1

10

1

0.1

100

10

1

0.1

(e)

5 10 15 20

100

10

1

0.1

1,00010

1

0.1

100

CAP0162 - adhE1CAP0163 - crfACAP0164 - ctfB

CAC2071 - spoA

CAC0323CAC0437CAC0903CAC2730CAC3319

CAC0225CAC0290CAC0863CAC1582CAC2434CAC3430

solexpression

solexpression

Kin

ase

expr

essi

on solexpressionsolexpression

(f) Time

6 10 18 26 34 44 66

Time (h)

sol operon

Orphan kinases

Two-component kinases

1 50 100Rank scale

Time (h)

Time (h)

Kin

ase

expr

essi

on

Kin

ase

expr

essi

on

Kin

ase

expr

essi

on

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.12

Expression profiles of sigma factors with unknown function and the effects of down-regulationFigure 6Expression profiles of sigma factors with unknown function and the effects of down-regulation. (a) Expression profiles of CAC3267 (open triangles), CAP0167 (filled squares), and CAP0157 (open circles) as ratios compared to the first expressed timepoint. Gray bar indicates the onset of transitional phase. (b) Expression profiles of CAC0550 (filled circles), CAC2052 (open squares), and CAC1766 (filled triangles) as ratios compared to the first expressed timepoint. Gray bar indicates the onset of transitional phase. (c) Ranked expression intensities of the sigma factors. White denotes a rank of 1, while dark blue denotes a rank of 100 (see scale). Gray squares indicate timepoints at which the intensity did not exceed the threshold value. (d) Microscopy time-course of asRNA strains compared to WT and plasmid control strains. Microscopy samples from WT (I) and pSOS95del (II) cultures (as controls) and three asRNA strains taken for two timepoints over a course of 72 hours. At 72 hours, WT (I) and pSOS95del (II) exhibit the typical clostridial forms (white arrows), while asCAP0166 (III) shows advanced differentiation with forespores and endospores (orange arrows) already visible. Strains asCAP0166 (III), asCAP0167 (IV), and asCAC1766 (V) show a novel, extra-swollen clostridial form (yellow arrows).

(a)

(b)

Exp

ress

ion

ratio

0 12 24 36 48 60

0 12 24 36 48 60

100

10

1

0.1

0.01

100

10

1

0.1

0.01

72 h48 h

CAC3267CAP0167CAP0157CAC0550CAC2052CAC1766

Exp

ress

ion

ratio

Time (h)

Time (h)

(c) Time

(d)

I

II

III

IV

V

I

II

III

IV

V1 50 100

Rank scale

6 10 18 26 34 44 66

Time (h)

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.13

sigma factors located on the pSOL1 plasmid as these may playa role in the regulation of sporulation. Two sigma factors,CAP0157 and CAP0167, are located on pSOL1 and are anno-tated as 'special sigma factor (σF/σE/σG family)' and 'special-ized sigma factor (σF/σE family)', respectively. It waspredicted that CAP0167 is putatively co-transcribed withCAP0166 from a promoter of the σF/σG family [37] and it dis-played an expression pattern similar to that of spo0A, consist-ent with the computational prediction of an 0A box [29] andtwo reverse 0A boxes in its promoter region (Figure 6a).CAP0157 was expressed from an unidentified promoter latein the timecourse (40+ hours) and thus may be involved inlate-stage sporulation, despite its low level of expression athour 20 (Figure 6a). CAC3267, putatively the fourth gene inan operon starting with CAC3270 and ending with CAC3264[37], was mainly expressed during early exponential growth(Figure 6a), then decreased, and peaked again around 14hours, after which expression decreased again. This patternof expression suggests that it plays a role in vegetative growthand possibly early sporulation. CAC0550, putatively tran-scribed from a σA promoter as a single cistron [37], wasmainly transcribed early with its expression ending after 20-24 hours (Figure 6b), suggesting that it is not involved insporulation. CAC1766, expressed from an unknown pro-moter, displayed a unique pattern with a progressive buildupstarting around hours 8-12 and a distinct peak around hour22 (Figure 6b). CAC2052 is annotated as 'DNA-dependentRNA polymerase σ-subunit' and was putatively expressedtogether with CAC2053, a hypothetical protein, from a σA

and/or a σF/σG promoter [37]. Our data suggest that it isunlikely to be transcribed from a σF/σG promoter without anyother effectors, as their transcription peaked at hour 16, whenthere was very little (if any) σF or σG activity (Figure 6b).

Phylogenetic tree comparisonTo help determine a possible function for these sigma factors,a phylogenetic tree was constructed of σ70 sigma factors fromten species, including B. subtilis and all sequenced clostridialspecies. The resulting tree (Additional data file 6) containseleven major branches, and of these, seven can be definitivelyclassified based on known sigma factors within the branch.These categories are extracytoplasmic function (ECF), sporu-lation factors (sigF, sigE, and sigG), sigH, sigA (a basal sigmafactor), sigD (regulates chemotaxis and motility), and sigB (ageneral response sigma factor). Two factors, CAC3267 andCAC1766, fell within ECF branches. CAC3267 fell within anECF branch close to the B. subtilis σV, a sigma factor ofunknown function, and σM, a sigma factor essential forgrowth and survival in high salt concentrations. CAC1766 fellwithin a different ECF branch close to B. subtilis σZ, a sigmafactor of unknown function, and CAC1509, a sigma factorexpressed for less than eight consecutive timepoints. Theremaining four factors fell within clusters with other clostrid-ial sigma factors of unknown function, though several couldhave possible ECF function.

Antisense RNA knock-down of four sigma factors: 'fat' clostridial forms and enhanced glucose metabolismOf the six expressed sigma factors of unknown function,CAP0157, CAP0167, CAC2052, and CAC1766 were chosen forfurther study because the timing and shape of their expres-sion patterns suggested potential involvement in sporulationand/or solventogenesis. Since the two processes are coupled,phenotypic changes in differentiation may affect solvent pro-duction, as has been previously observed [4,6,29,33,49].Antisense RNA (asRNA) knock-down was chosen over knock-ing out the genes, because knockouts are still extremelydifficult to produce in this and all other clostridia. Indeed, todate, only a handful of knockouts have been created [29,50-53], and these have only been achieved after screening thou-sands of transformants [51-53]. Recently, a group II intronsystem has been developed for clostridia [54], but this systemwas not yet available when these experiments were carriedout. In contrast, asRNA is relatively quick, has been shown toreduce gene expression by up to 90% [33,55,56] and has beenused to knock-down a large number of genes with a high levelof specificity [33,49,55-59]. asRNA constructs (see Additionaldata file 7 for specific sequences used) were designed againstCAP0157, CAP0167, CAC2052, and CAC1766 along withCAC2053 and CAP0166, the first genes in the operonspredicted to contain CAC2052 and CAP0167, respectively[37]. Cultures of these strains were examined and comparedagainst the wild type (WT) and plasmid control strain824(pSOS95del) for cell morphology differences and meta-bolic changes.

Microscopy results from the asRNA-strain cultures revealedboth novel morphologies and apparently altered differentia-tion (Figure 6d). Most notable were changes in strainsasCAP0166, asCAP0167 and asCAC1766. Typical WT culturesdisplay a predominately vegetative, symmetrically dividingpopulation through 72 hours as evidenced by the thin, rod-shaped, phase dark cells (Figure 6d, I). By 72 hours, WT cul-tures exhibited only a small percentage of swollen, cigar-shaped clostridial forms and then a proportional populationof free spores by 96 hours.

pSOS95del cultures exhibited clostridial forms by 48 hours,suggesting an accelerated differentiation compared to WT, ashas been seen before in our laboratory (Figure 6d, II). More-over, a greater percentage of clostridial forms and free sporescompared to WT were observed at 72 and 96 hours, respec-tively. asCAP0166 cultures generated a large percentage ofclostridial forms and endospores/free spores by hours 48 and72, respectively (Figure 6d, III). This differentiation is accel-erated in comparison to pSOS95del. By hour 96, asCAP0166cultures exhibited predominately vegetative cells apparentlyderived from germinated spores (data not shown).asCAP0167 cultures also exhibited accelerated differentiationand displayed a novel (to our knowledge) form of cellularmorphology that was most profoundly observable at 72 hours(Figure 6d, IV). This novel morphology has qualities of an

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.14

excessively swollen clostridial cigar-form (which makes themlook much shorter than normal clostridial forms), with whatappears to be endospore formation occurring, but without theassociated phase bright characteristics seen in the 72 hourasCAP0166 cultures. The asCAP0166 culture displayed cellsin this novel morphological state as well, but to a lesserextent, although it is possible that because of its faster sporu-lation, such cell forms appeared prior to 72 hours. TheasCAC1766 cultures also exhibited altered differentiation;most importantly, at 72 hours the majority of the cells exhib-ited a very swollen clostridial-form morphology similar tothat in the asCAP0167 cultures at 72 hours, but slightly moreelongated (Figure 6d, V).

To further characterize this novel cell form, transmissionelectron microscopy (TEM) and scanning electron micros-copy images of cells were taken for strains asCAP0167 andasCAC1766. To determine morphological differencesinvolved in differentiation, the TEM images were comparedagainst cell images taken from the plasmid control strain(Figure 7). For both asRNA strains, the very swollen cellforms observed can be documented as approximately 2.5-4μm long, and 1.1-1.3 μm in diameter, and should be comparedto control or WT swollen clostridial forms, which are 3.5-6μm long and 0.8-1 μm in diameter. Forespore and endosporeforms of both asCAP0167 (Figure 7c,d) and asCAC1766(Figure 7e,f) displayed a pinched end not seen in the plasmidcontrol (Figure 7b). A slight pinching is seen in the clostridialforms of the plasmid control strain (Figure 7a), but this isprobably indicative that an asymmetric division is about tooccur. Rather, the pinched ends seen in the antisense strainsoccur after asymmetric division and while the spore is devel-oping within the mother cell. These pinched ends are alsonoticeable in the scanning electron microscopy images (Fig-ure 8). Though granulose is distinguishable in most of theTEM images (Figure 7c,d,f), it is not the characteristic elec-tron translucent seen in typical clostridial, forespore, andendospore forms (Figure 7a,b). These differences were seenthroughout the culture and additional TEM images of boththe plasmid control and the antisense strains are included inAdditional data file 8.

Glucose, acetone, and butanol concentrations from two tofour biological replicates for each strain were averagedtogether, and the results are shown in Table 1. We averageddata from cultures that displayed similar characteristics;most cultures did so despite the fact that each culture wasinoculated from a different colony for each strain. Acetoneand butanol levels were typical for WT and control cultures,with the WT producing 90 mM of acetone and 150 mM ofbutanol and the plasmid-control strain producing 80 mM ofacetone and 160 mM of butanol [60]. By 192 hours, all strainshad either produced comparable amounts of butanol to theWT and the plasmid control strain or had somewhat outper-formed these two strains. The most significant differenceswere that all asRNA strains consumed higher levels of glucose

and also had a delayed metabolism in terms of product forma-tion. These metabolic changes, although preliminary, areconsistent with and support the large changes in the kineticsof sporulation observed by microscopy.

ConclusionThis detailed and previously unrevealed transcriptional road-map has allowed for the first time a complete investigation ofthe genetic events associated with clostridial differentiation.We were able to link distinct and striking globaltranscriptional changes to previously known important mor-phological and physiological changes. To date, this is the mostcomplete genetic analysis of the different morphologicalforms: vegetative, clostridial, and forespore/endospore.Importantly, this analysis was performed on a mixed culture,which may either dilute or produce noise in the data, butinvestigation of the clusters identified revealed that theseclusters do capture important known processes. We were alsoable to identify a cell population late in the timecourse similarto vegetative cells. Visually, these late cells looked and actedlike vegetative cells, and transcriptionally, they were alsofairly similar. The major cell motility and chemotaxis geneswere upregulated both early and late in the timecourse(Figure S2 in Additional data file 3), as were the ribosomalproteins (Figure S12 in Additional data file 3). Also, the celldivision associated genes rodA, ftsE, and ftsX follow the sametranscriptional pattern of both early and late expression (Fig-ure S11 in Additional data file 3). Although, these cells staindifferently from the early vegetative cells, probably due tochanges in membrane structure in response to the presence ofsolvents and do not produce detectable levels of acids or sol-vents, we believe these cells are germinated cells from sporesproduced early in the timecourse. While the triggers for bothsporulation and germination are not known [1], the culturelate in the timecourse is less acidic because of the acid reas-similation, and pH has been shown to be a trigger for sporu-lation [21].

This study has also allowed the first full comparison to thewidely studied B. subtilis sporulation program. We have con-fidently identified the temporal orchestration of all knownsporulation-related transcription factors and conclude theBacillus model generally holds true with the cascade pro-gressing in the following manner: σH, Spo0A, σF, σE, and σG

(Figure 4f). In addition, we can conclude that the major acti-vating/processing proteins involved in sigma factor activa-tion in B. subtilis play a similar role in C. acetobutylicum,though additional investigation is needed to clarify their role.Of significance is the lack of sigK signal. The genes responsi-ble for transcribing sigK in B. subtilis, sigE and spoIIID, wereexpressed, but the putative processing enzyme spoIVFB wasnot. Two genes under the control of σK in B. subtilis wereexpressed, but their expression patterns are not consistentwith each other. Based on the expression pattern of yabG, it

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.15

Figure 7 TEM images of the novel cell forms. (a-b) TEM images of the plasmid control strain pSOS95del: typical elongated clostridial form with electron translucent granulose (a); typical endospore form with a developing endospore at one end of the cell and electron translucent granulose still visible at the other end of the cell (b). (c-d) TEM images of the antisense strain asCAP0167. (e-f) TEM images of the antisense strain asCAC1766. Red arrows in (c-f) indicate pinched portions of the cell membrane not seen in the control strain and are characteristic of this novel cell type. Also noticeable is the electron dense granulose in the antisense strains, in contrast to the electron translucent granulose in the control samples.

(a)

(c)

(b)

(e)

(d)

(f)

1,000 nm 1,000 nm

1,000 nm 1,000 nm

1,000 nm 1,000 nm

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.16

could be controlled by σE, while the late expression of spsFcould be an indication of σK activity.

Finally, in order to determine if one of the annotated sigmafactors of unknown function could be a sigK-like gene, wefirst investigated their transcriptional profiles. CAP0157 wasa possible candidate with its upregulation late in the time-course, as was CAC1766 since its expression was sustainedthroughout the stationary phase (Figure 6a,b). Neither ofthese genes, nor any of the other sigma factors of unknownfunction, clustered close to the known sporulation-relatedsigma factors on the phylogenetic tree (Additional data file 6),but when downregulated using asRNA, both CAC1766 and theCAP0167 operon (CAP0166 and CAP0167) displayed altereddifferentiation (Figures 6d, 7 and 8). Though involved in dif-ferentiation, the exact role of these two sigma factors is diffi-cult to assess because of the incomplete silencing of the genesthrough asRNA downregulation. Mature free spores and typ-ical endospore forms without a pinched end are still seen(data not shown), but whether these develop from the novelcell types or from cells not affected by the antisense cannot bedetermined. Interestingly, both CAP0167 and CAC1766 clus-tered together with other clostridial sigma factors and closerto ECF sigma factors than to the major sporulation sigma fac-tors sigF, sigE, and sigG (Additional data file 6). In B. subtilis,ECF sigma factors do not play a role in differentiation [61,62],though a triple mutant in sigM, sigW, and sigX did displayaltered phenotypes [62]. The fact that CAC1766 and CAP0167appear to affect the developmental process of sporulation(Figures 7 and 8; Additional data file 8) suggests either thatECF factors may play a role in sporulation in clostridia or thata novel category of sigma factors exist in clostridia that play arole in sporulation.

Materials and methodsFermentation analysisTwo cultures of C. acetobutylicum ATCC 824 were grown inpH controlled (pH >5) bioreactors (Bioflow II and 110, NewBrunswick Scientific, Edison, NJ, USA) [7]. Cell density, sub-strate and product concentrations were analyzed as described[56].

RNA isolation and cDNA labelingSamples were collected by centrifuging 3-10 ml of culture at5,000×g for 10 minutes, 4°C and storing the cell pellets at -85°C. Prior to RNA isolation, cells were washed in 1 ml SETbuffer (25% sucrose, 50 mM EDTA [pH 8.0], and 50 mMTris-HCl [pH 8.0]) and centrifuged at 5,000×g for 10 min-utes, 4°C. Pellets were processed similarly to [7] but with thenoted modifications. Cells were lysed by resuspending in 220μl SET buffer with 20 mg/ml lysozyme (Sigma, St. Louis, MO,USA) and 4.55 U/ml proteinase K (Roche, Indianapolis, IN,USA) and incubated at room temperature for 6 minutes. Fol-lowing incubation, 40 mg of acid-washed glass beads (≤106μm; Sigma) were added to the solution, and the mixture wascontinuously vortexed for 4 minutes at room temperature.Immediately afterwards, 1 ml of ice cold TRIzol (Invitrogen,Carlsbad, CA, USA) was added; 500 μl of sample was dilutedwith an equal volume of ice cold TRIzol and purified. Follow-ing dilution, 200 μl of ice cold chloroform was added to eachsample, mixed vigorously for 15 s, and incubated at roomtemperature for 3 minutes. Samples were then centrifuged at12,000 rpm in a tabletop microcentrifuge for 15 minutes at4°C. The upper phase was saved and diluted by adding 500 μlof 70% ethanol. Samples were then applied to the RNeasyMini Kit (Qiagen, Valencia, CA, USA), following the manufac-turer's instructions. To minimize genomic DNA contamina-tion, samples were incubated with the RW1 buffer at roomtemperature for 4 minutes. The method disrupted all celltypes equally, as evidenced by microscopy (data not shown).cDNA was generated and labeled as described [7]. The refer-

Table 1

Concentrations of glucose, acetone, and butanol for asRNA strains

96 hours 144 hours 192 hours*

Sample Glucose† Acetone† Butanol† Glucose† Acetone† Butanol† Glucose† Acetone† Butanol†

Wild type 165 91 157 143 74 157 120 61 162

pSOS95del‡ 264 57 97 136 83 169 125 57 158

asCAC1766 274 67 84 118 123 169 114 97 163

asCAC2052 294 49 69 191 84 122 116 92 154

asCAC2053 285 54 77 158 94 142 94 88 161

asCAP0157 314 49 63 198 91 122 96 111 174

asCAP0166 290 55 77 118 125 167 77 91 176

asCAP0167 294 54 73 78 125 180 56 98 185

*At 192 hours, significant amounts of acetone had evaporated along with small amounts of butanol. However, the cultures were still metabolically active, as indicated by the decreased amounts of glucose and increased amounts of butanol. †Concentrations are mM. ‡pSOS95del was used as a plasmid control strain.

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.17

ence RNA pool contained 25 μg of RNA from samples takenfrom the same culture at 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 44, 48, 54, 58, and 66 h.

Microarray analysisAgilent technology 22k arrays, (GEO accession numberGPL4412) as described in [63], were hybridized, washed, andscanned per Agilent's recommendations. Spot quantificationemployed Agilent's eXtended Dynamic Range technique with

Scanning electron microscopy (SEM) images of the novel cell formsFigure 8Scanning electron microscopy (SEM) images of the novel cell forms. SEM images of the antisense strains (a-c) asCAP0167 and (d-f) asCAC1766. Red arrows in indicate pinched portions of the cell membrane not seen in the control strain and are characteristic of this novel cell type.

(a)

(c)

(b)

(e)

(d)

(f)

2.50 µm 5.00 µm

2.50 µm 2.50 µm

2.50 µm 2.50 µm

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.18

gains of 100% and 10% (Agilent's Feature Extraction software(v. 9.1)). Normalization and slide averaging was carried out asdescribed [7,63]. A minimum intensity of 50 intensity unitswas used as described [63]. Microarray data have been depos-ited in the Gene Expression Omnibus database under acces-sion number GSE6094. To gain a qualitative measure of theabundance of an mRNA transcript, the averaged normalizedlog mean intensity values were ranked on a scale of 1 (lowestintensity value) to 100 (highest intensity value). Genes wereclustered using TIGR's MEV program [64].

Quantitative RT-PCRQ-RT-PCR was performed as described [48]. Specific primersequences are included in Additional data file 9; CAC3571 wasused as the housekeeping gene.

MicroscopyFor light microscopy, samples were stored at -85°C after 15%glycerol was added to the sampled culture. Samples were thenpelleted, washed twice with 1% w/v NaCl and fixed using 50μl of 0.05% HCl/0.5% NaCl solution to a final count of 106

cells/μl. Slides were imaged using a Leica widefieldmicroscope with either phase contrast or Syto-9 and PI dyes(Invitrogen LIVE/DEAD BacLight Kit) to distinguish cellmorphology.

For electron microscopy, samples were fixed by addition of16% paraformaldehyde and 8% glutaraldehyde to the culturemedium for a final concentration of 2% paraformaldehydeand 2% glutaraldehyde. For cultures grown on plates, colo-nies were scraped from the agar and suspended in 2% para-formaldehyde and 2% glutaraldehyde in 0.1 M sodiumcacodylate buffer (pH 7.4). Cultures were fixed for 1 h at roomtemperature, pelleted and resuspended in buffer.

For transmission electron microscopy, bacteria were pelleted,embedded in 4% agar and cut into 1 mm × 1 mm cubes. Thesamples were washed three times for 15 minutes in 0.1 Msodium cacodylate buffer (pH 7.4), fixed in 1% osmiumtetroxide in buffer for 2 h, and then washed extensively withbuffer and double de-ionized water. Following dehydration inan ascending series of ethanol (25, 50, 75, 95, 100, 100%; 15minutes each), the samples were infiltrated with Embed-812resin in 100% ethanol (1:3, 1:2, 1:1, 2:1, 3:1; 1 h each) and thenseveral changes in 100% resin. After an overnight infiltrationin 100% resin, the samples were embedded in BEEM capsulesand polymerized at 65°C for 48 h. Blocks were sectioned on aReichert-Jung UltracutE ultramicrotome and ultrathin sec-tions were collected onto formvar-carbon coated coppergrids. Sections were stained with methanolic uranyl acetateand Reynolds' lead citrate [65] and viewed on a Zeiss CEM902 transmission electron microscope at 80 kV. Images wererecorded with an Olympus Soft Imaging System GmbH Meg-aview II digital camera. Brightness levels were adjusted in theimages so that the background between images appearedsimilar.

For scanning electron microscopy, fixed samples were incu-bated on poly-L-lysine coated silica wafers for 1 h and thenrinsed three times for 15 minutes in 0.1 M sodium cacodylatebuffer (pH 7.4). The samples were fixed with 1% osmiumtetroxide in buffer for 2 h, washed in buffer and double de-ionized water, and then dehydrated in ethanol (25, 50, 75, 95,100, 100%; 15 minutes each). The wafers were critical pointdried in an Autosamdri 815B critical point drier and mountedonto aluminum stubs with silver paint. The samples werecoated with Au/Pd with a Denton Bench Top Turbo III sput-ter-coater and viewed with a Hitachi 4700 FESEM at 3.0 kV.

Phylogenetic tree generationBased on the genome annotations available at NCBI, we con-sidered any sigma factor that was annotated as σ70 or unanno-tated. A second filter was applied by requiring that all thesequences should contain a Region 2, the most conservedregion of the σ70 protein. All members of this class of sigmafactor contain Region 2, and it was modeled with the HMMpfam04542. This criterion removed CAC0550, CAC1766 andCAP0157, but they were added to the list again despite theirlack of a Region 2. The alignment was made using ClustalW1.83 using the default settings and visualized as a radial treeas created by Phylodraw v. 0.8 from Pusan NationalUniversity.

Generation and characterization of antisense strainsOligonucleotides were designed to produce asRNA comple-mentary to the upstream 20 bp and first 30-40 bp of the tar-geted genes' transcripts (Additional data file 7). Theconstructs were cloned into pSOS95del under the control of athiolase (thl) promoter and confirmed by restriction digest.Plasmids were then methylated and transformed into C. ace-tobutylicum ATCC 824, as previously described [33,55,56].Strains were grown in 10 ml cultures and characterized usingmicroscopy and HPLC to analyze final product concentra-tions [56].

AbbreviationsasRNA, antisense RNA; COG, Cluster of Orthologous Groups;ECF, extracytoplasmic function; PI, propidium iodide; Q-RT-PCR, quantitative reverse transcription PCR; TEM, transmis-sion electron microscopy; WT, wild type.

Authors' contributionsSWJ carried out the microarray experiments, helped with theelectron microscopy, helped analyze the data, and draftedand finalized the manuscript. CJP designed the microarrayplatform used, helped with the bioinformatic tools used in theanalysis, and drafted parts of the manuscript. BT carried outall the microscopy except the electron microscopy and gener-ated the antisense RNA strains. NC carried out the microar-ray experiments and helped with the generation of theantisense strains. RS helped design the microarray experi-

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.19

ments, carried out the Q-RT-PCR experiments, helped ana-lyze the data, and drafted parts of the manuscript. RSS helpedwith the bioinformatic tools used in the analysis. ETP helpedin the design of all the experiments, the analysis and interpre-tation of the data, and helped in the organization, draft andediting of the manuscript. All authors read and approved thefinal manuscript.

Additional data filesThe following additional data are available. Additional datafile 1 is a figure comparing the present microarray study to anearlier microarray study that examined the early sporulationof C. acetobutylicum followed by a brief discussion. Addi-tional data file 2 contains tables detailing the COG analysis foreach cluster and all the genes placed in each cluster. Addi-tional data file 3 contains figures of the transcriptional pro-files, in terms of both intensity and differential expression, ofspecific gene clusters with brief discussions following severalfigures. Additional data file 4 is a composite figure showingthe individual expression profiles of the genes that werestandardized and averaged and is followed by a briefdiscussion on how the genes used to construct the deducedactivity plots were chosen. Additional data file 5 is a figureshowing the differential expression and intensity of all anno-tated histidine kinases and response regulators. Additionaldata file 6 is a figure showing the phylogenetic tree resultingfrom the alignment of the σ70-related and unannotated sigmafactors from ten bacterial species. Additional data file 7 is atable listing the sequences for each asRNA construct. Addi-tional data file 8 contains figures showing additional TEMimages of the plasmid control strain, asCAP0167, andasCAC1766. Additional data file 9 is a table listing the primersequences used in the Q-RT-PCR experiments.Additional data file 1Comparison of the present microarray study to an earlier microar-ray study that examined the early sporulation of C. acetobutylicumComparison of the present microarray study to an earlier microar-ray study that examined the early sporulation of C. acetobutylicum.Click here for fileAdditional data file 2COG analysis for each cluster and all the genes placed in each clusterCOG analysis for each cluster and all the genes placed in each cluster.Click here for fileAdditional data file 3Transcriptional profiles, in terms of both intensity and differential expression, of specific gene clustersTranscriptional profiles, in terms of both intensity and differential expression, of specific gene clusters.Click here for fileAdditional data file 4Profiles of the genes that were standardized and averagedIncludes a brief discussion on how the genes used to construct the deduced activity plots were chosen.Click here for fileAdditional data file 5Differential expression and intensity of all annotated histidine kinases and response regulatorsDifferential expression and intensity of all annotated histidine kinases and response regulators.Click here for fileAdditional data file 6Phylogenetic tree resulting from the alignment of the σ70-related and unannotated sigma factors from ten bacterial speciesPhylogenetic tree resulting from the alignment of the σ70-related and unannotated sigma factors from ten bacterial species.Click here for fileAdditional data file 7Sequences for each asRNA constructSequences for each asRNA construct.Click here for fileAdditional data file 8TEM images of the plasmid control strain, asCAP0167, and asCAC1766TEM images of the plasmid control strain, asCAP0167, and asCAC1766.Click here for fileAdditional data file 9Primer sequences used in the Q-RT-PCR experimentsPrimer sequences used in the Q-RT-PCR experiments.Click here for file

AcknowledgementsWe acknowledge the use of the Northwestern University Keck BiophysicsFacility, the Northwestern University Biological Imaging Facility for the lightmicroscopy, and Shannon Modla in the Delaware Biotechnology InstituteBio-Imaging Facility for the electron microscopy. Supported by NSF grant(BES-0418157) and an NIH/NIGMS Biotechnology Training grant (T32-GM08449) fellowship for Bryan Tracy.

References1. Paredes CJ, Alsaker KV, Papoutsakis ET: A comparative genomic

view of clostridial sporulation and physiology. Nat Rev Microbiol2005, 3:969-978.

2. Demain AL, Newcomb M, Wu JH: Cellulase, clostridia, andethanol. Microbiol Mol Biol Rev 2005, 69:124-154.

3. Woods DR: The genetic engineering of microbial solventproduction. Trends Biotechnol 1995, 13:259-264.

4. Alsaker KV, Spitzer TR, Papoutsakis ET: Transcriptional analysisof spo0A overexpression in Clostridium acetobutylicum and itseffect on the cell's response to butanol stress. J Bacteriol 2004,186:1959-1971.

5. Tomas CA, Beamish J, Papoutsakis ET: Transcriptional analysis ofbutanol stress and tolerance in Clostridium acetobutylicum. JBacteriol 2004, 186:2006-2018.

6. Zhao Y, Tomas CA, Rudolph FB, Papoutsakis ET, Bennett GN: Intra-cellular butyryl phosphate and acetyl phosphate concentra-

tions in Clostridium acetobutylicum and their implications forsolvent formation. Appl Environ Microbiol 2005, 71:530-537.

7. Alsaker KV, Papoutsakis ET: Transcriptional program of earlysporulation and stationary-phase events in Clostridiumacetobutylicum. J Bacteriol 2005, 187:7103-7118.

8. Jones DT, Westhuizen A van der, Long S, Allcock ER, Reid SJ, WoodsDR: Solvent production and morphological changes inClostridium acetobutylicum. Appl Environ Microbiol 1982,43:1434-1439.

9. Long S, Jones DT, Woods DR: Sporulation of Clostridium aceto-butylicum P262 in a defined medium. Appl Environ Microbiol 1983,45:1389-1393.

10. Comas-Riu J, Vives-Rego J: Cytometric monitoring of growth,sporogenesis and spore cell sorting in Paenibacillus polymyxa(formerly Bacillus polymyxa). J Appl Microbiol 2002, 92:475-481.

11. Yang H, Haddad H, Tomas C, Alsaker K, Papoutsakis ET: A segmen-tal nearest neighbor normalization and gene identificationmethod gives superior results for DNA-array analysis. ProcNatl Acad Sci USA 2003, 100:1122-1127.

12. Tatusov RL, Galperin MY, Natale DA, Koonin EV: The COG data-base: a tool for genome-scale analysis of protein functionsand evolution. Nucleic Acids Res 2000, 28:33-36.

13. Nölling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, GibsonR, Lee HM, Dubois J, Qiu D, Hitti J, GTC Sequencing Center Produc-tion, Finishing, and Bioinformatics Teams, Wolf YI, Tatusov RL,Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN,Koonin EV, Smith DR: Genome sequence and comparativeanalysis of the solvent-producing bacterium Clostridiumacetobutylicum. J Bacteriol 2001, 183:4823-4838.

14. Lyristis M, Boynton ZL, Petersen D, Kan Z, Bennett GN, Rudolph FB:Cloning, sequencing, and characterization of the geneencoding flagellin, flaC, and the post-translational modifica-tion of flagellin, FlaC, from Clostridium acetobutylicumATCC824. Anaerobe 2000, 6:69-79.

15. Welch M, Oosawa K, Aizawa SI, Eisenbach M: Effects of phospho-rylation, Mg2+, and conformation of the chemotaxis proteinCheY on its binding to the flagellar switch protein FliM. Bio-chemistry 1994, 33:10470-10476.

16. Baer SH, Blaschek HP, Smith TL: Effect of butanol challenge andtemperature on lipid composition and membrane fluidity ofbutanol-tolerant Clostridium acetobutylicum. Appl EnvironMicrobiol 1987, 53:2854-2861.

17. Lepage C, Fayolle F, Hermann M, Vandecasteele J-P: Changes inmembrane lipid composition of Clostridium acetobutylicumduring acetone-butanol fermentation: effects of solvents,growth temperature and pH. J Gen Microbiol 1987, 133:103-110.

18. Vollherbst-Schneck K, Sands JA, Montenecourt BS: Effect of buta-nol on lipid composition and fluidity of Clostridium aceto-butylicum ATCC 824. Appl Environ Microbiol 1984, 47:193-194.

19. Zhao Y, Hindorff LA, Chuang A, Monroe-Augustus M, Lyristis M, Har-rison ML, Rudolph FB, Bennett GN: Expression of a cloned cyclo-propane fatty acid synthase gene reduces solvent formationin Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol2003, 69:2831-2841.

20. Peguin S, Soucaille P: Modulation of carbon and electron flow inClostridium acetobutylicum by iron limitation and methyl vio-logen addition. Appl Environ Microbiol 1995, 61:403-405.

21. Jones DT, Woods DR: Acetone-butanol fermentationrevisited. Microbiol Rev 1986, 50:484-524.

22. Cornillot E, Nair RV, Papoutsakis ET, Soucaille P: The genes forbutanol and acetone formation in Clostridium acetobutylicumATCC 824 reside on a large plasmid whose loss leads todegeneration of the strain. J Bacteriol 1997, 179:5442-5447.

23. Schaffer S, Isci N, Zickner B, Dürre P: Changes in proteinsynthesis and identification of proteins specifically inducedduring solventogenesis in Clostridium acetobutylicum. Electro-phoresis 2002, 23:110-121.

24. Mansilla MC, Cybulski LE, Albanesi D, de Mendoza D: Control ofmembrane lipid fluidity by molecular thermosensors. JBacteriol 2004, 186:6681-6688.

25. Kaan T, Homuth G, Mader U, Bandow J, Schweder T: Genome-wide transcriptional profiling of the Bacillus subtilis cold-shock response. Microbiology 2002, 148:3441-3455.

26. Johnston NC, Goldfine H: Lipid composition in the classificationof the butyric acid-producing clostridia. J Gen Microbiol 1983,129:1075-1081.

27. Burbulys D, Trach KA, Hoch JA: Initiation of sporulation in B.subtilis is controlled by a multicomponent phosphorelay. Cell

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.20

1991, 64:545-552.28. Stragier P, Losick R: Molecular genetics of sporulation in Bacil-

lus subtilis. Annu Rev Genet 1996, 30:297-241.29. Harris LM, Welker NE, Papoutsakis ET: Northern, morphological,

and fermentation analysis of spo0A inactivation and overex-pression in Clostridium acetobutylicum ATCC 824. J Bacteriol2002, 184:3586-3597.

30. Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, LiuJS, Losick R: The Spo0A regulon of Bacillus subtilis. Mol Microbiol2003, 50:1683-1701.

31. Cervin MA, Lewis RJ, Brannigan JA, Spiegelman GB: The Bacillussubtilis regulator SinR inhibits spoIIG promoter transcriptionin vitro without displacing RNA polymerase. Nucleic Acids Res1998, 26:3806-3812.

32. Mandic-Mulec I, Doukhan L, Smith I: The Bacillus subtilis SinR pro-tein is a repressor of the key sporulation gene spo0A. JBacteriol 1995, 177:4619-4627.

33. Scotcher MC, Rudolph FB, Bennett GN: Expression of abrB310and sinR, and effects of decreased abrB310 expression on thetransition from acidogenesis to solventogenesis, in Clostrid-ium acetobutylicum ATCC 824. Appl Environ Microbiol 2005,71:1987-1995.

34. Perego M, Spiegelman GB, Hoch JA: Structure of the gene for thetransition state regulator, abrB: regulator synthesis is con-trolled by the spo0A sporulation gene in Bacillus subtilis. MolMicrobiol 1988, 2:689-699.

35. Chary VK, Meloni M, Hilbert DW, Piggot PJ: Control of the expres-sion and compartmentalization of (sigma)G activity duringsporulation of Bacillus subtilis by regulators of (sigma)F and(sigma)E. J Bacteriol 2005, 187:6832-6840.

36. Sun DX, Cabrera-Martinez RM, Setlow P: Control of transcriptionof the Bacillus subtilis spoIIIG gene, which codes for the fore-spore-specific transcription factor sigma G. J Bacteriol 1991,173:2977-2984.

37. Paredes CJ, Rigoutsos I, Papoutsakis ET: Transcriptional organiza-tion of the Clostridium acetobutylicum genome. Nucleic AcidsRes 2004, 32:1973-1981.

38. Kroos L, Kunkel B, Losick R: Switch protein alters specificity ofRNA polymerase containing a compartment-specific sigmafactor. Science 1989, 243:526-529.

39. Stragier P, Kunkel B, Kroos L, Losick R: Chromosomal rearrange-ment generating a composite gene for a developmentaltranscription factor. Science 1989, 243:507-512.

40. Sauer U, Treuner A, Buchholz M, Santangelo JD, Dürre P: Sporula-tion and primary sigma factor homologous genes in Clostrid-ium acetobutylicum. J Bacteriol 1994, 176:6572-6582.

41. Santangelo JD, Kuhn A, Treuner-Lange A, Dürre P: Sporulation andtime course expression of sigma-factor homologous genes inClostridium acetobutylicum. FEMS Microbiol Lett 1998,161:157-164.

42. Tatti KM, Jones CH, Moran CP Jr: Genetic evidence for interac-tion of sigma E with the spoIIID promoter in Bacillus subtilis.J Bacteriol 1991, 173:7828-7833.

43. Piggot PJ, Hilbert DW: Sporulation of Bacillus subtilis. Curr OpinMicrobiol 2004, 7:579-586.

44. Wörner K, Szurmant H, Chiang C, Hoch JA: Phosphorylation andfunctional analysis of the sporulation initiation factor Spo0Afrom Clostridium botulinum. Mol Microbiol 2006, 59:1000-1012.

45. Jiang M, Shao W, Perego M, Hoch JA: Multiple histidine kinasesregulate entry into stationary phase and sporulation in Bacil-lus subtilis. Mol Microbiol 2000, 38:535-542.

46. Dartois V, Djavakhishvili T, Hoch JA: Identification of a mem-brane protein involved in activation of the KinB pathway tosporulation in Bacillus subtilis. J Bacteriol 1996, 178:1178-1186.

47. LeDeaux JR, Grossman AD: Isolation and characterization ofkinC, a gene that encodes a sensor kinase homologous to thesporulation sensor kinases KinA and KinB in Bacillus subtilis.J Bacteriol 1995, 177:166-175.

48. Alsaker KV, Paredes CJ, Papoutsakis ET: Design, optimization andvalidation of genomic DNA microarrays for examining theClostridium acetobutylicum transcriptome. Biotechnol BioprocessEng 2005, 10:432-443.

49. Scotcher MC, Bennett GN: SpoIIE regulates sporulation butdoes not directly affect solventogenesis in Clostridium aceto-butylicum ATCC 824. J Bacteriol 2005, 187:1930-1936.

50. Green EM, Boynton ZL, Harris LM, Rudolph FB, Papoutsakis ET, Ben-nett GN: Genetic manipulation of acid formation pathways bygene inactivation in Clostridium acetobutylicum ATCC 824.

Microbiology 1996, 142:2079-2086.51. Huang IH, Waters M, Grau RR, Sarker MR: Disruption of the gene

(spo0A) encoding sporulation transcription factor blocksendospore formation and enterotoxin production in entero-toxigenic Clostridium perfringens type A. FEMS Microbiol Lett2004, 233:233-240.

52. Raju D, Waters M, Setlow P, Sarker MR: Investigating the role ofsmall, acid-soluble spore proteins (SASPs) in the resistanceof Clostridium perfringens spores to heat. BMC Microbiol 2006,6:50.

53. Sarker MR, Carman RJ, McClane BA: Inactivation of the gene(cpe) encoding Clostridium perfringens enterotoxin elimi-nates the ability of two cpe-positive C. perfringens type Ahuman gastrointestinal disease isolates to affect rabbit ilealloops. Mol Microbiol 1999, 33:946-958.

54. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP: TheClosTron: a universal gene knock-out system for the genusClostridium. J Microbiol Methods 2007, 70:452-464.

55. Desai RP, Papoutsakis ET: Antisense RNA strategies for meta-bolic engineering of Clostridium acetobutylicum. Appl EnvironMicrobiol 1999, 65:936-945.

56. Tummala SB, Welker NE, Papoutsakis ET: Design of antisenseRNA constructs for downregulation of the acetone forma-tion pathway of Clostridium acetobutylicum. J Bacteriol 2003,185:1923-1934.

57. Perret S, Maamar H, Belaich JP, Tardif C: Use of antisense RNA tomodify the composition of cellulosomes produced byClostridium cellulolyticum. Mol Microbiol 2004, 51:599-607.

58. Raju D, Setlow P, Sarker MR: Antisense-RNA-mediateddecreased synthesis of small, acid-soluble spore proteinsleads to decreased resistance of Clostridium perfringensspores to moist heat and UV radiation. Appl Environ Microbiol2007, 73:2048-2053.

59. Tummala SB, Junne SG, Papoutsakis ET: Antisense RNA downreg-ulation of coenzyme A transferase combined with alcohol-aldehyde dehydrogenase overexpression leads to predomi-nantly alcohologenic Clostridium acetobutylicum fermenta-tions. J Bacteriol 2003, 185:3644-3653.

60. Tomas CA, Welker NE, Papoutsakis ET: Overexpression ofgroESL in Clostridium acetobutylicum results in increased sol-vent production and tolerance, prolonged metabolism, andchanges in the cell's transcriptional program. Appl EnvironMicrobiol 2003, 69:4951-4965.

61. Asai K, Ishiwata K, Matsuzaki K, Sadaie Y: A viable Bacillus subtilisstrain without functional extracytoplasmic function sigmagenes. J Bacteriol 2008, 190:2633-2636.

62. Mascher T, Hachmann AB, Helmann JD: Regulatory overlap andfunctional redundancy among Bacillus subtilis extracytoplas-mic function sigma factors. J Bacteriol 2007, 189:6919-6927.

63. Paredes CJ, Senger RS, Spath IS, Borden JR, Sillers R, Papoutsakis ET:A general framework for designing and validating oligomer-based DNA microarrays and its application to Clostridiumacetobutylicum. Appl Environ Microbiol 2007, 73:4631-4638.

64. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J,Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A,Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A,Trush V, Quackenbush J: TM4: a free, open-source system formicroarray data management and analysis. Biotechniques 2003,34:374-378.

65. Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 1963,17:208-212.

66. Frey M: Hydrogenases: hydrogen-activating enzymes. Chembi-ochem 2002, 3:153-160.

67. Gorwa MF, Croux C, Soucaille P: Molecular characterization andtranscriptional analysis of the putative hydrogenase gene ofClostridium acetobutylicum ATCC 824. J Bacteriol 1996,178:2668-2675.

68. Moir A, Corfe BM, Behravan J: Spore germination. Cell Mol Life Sci2002, 59:403-409.

69. Igarashi T, Setlow P: Transcription of the Bacillus subtilis gerKoperon, which encodes a spore germinant receptor, andcomparison with that of operons encoding other germinantreceptors. J Bacteriol 2006, 188:4131-4136.

70. Dürre P, Hollergschwandner C: Initiation of endospore forma-tion in Clostridium acetobutylicum. Anaerobe 2004, 10:69-74.

71. Makino S, Moriyama R: Hydrolysis of cortex peptidoglycan dur-ing bacterial spore germination. Med Sci Monit 2002,

Genome Biology 2008, 9:R114

http://genomebiology.com/2008/9/7/R114 Genome Biology 2008, Volume 9, Issue 7, Article R114 Jones et al. R114.21

8:RA119-127.72. Ishikawa S, Yamane K, Sekiguchi J: Regulation and characteriza-

tion of a newly deduced cell wall hydrolase gene (cwlJ) whichaffects germination of Bacillus subtilis spores. J Bacteriol 1998,180:1375-1380.

73. Kodama T, Takamatsu H, Asai K, Kobayashi K, Ogasawara N, WatabeK: The Bacillus subtilis yaaH gene is transcribed by SigE RNApolymerase during sporulation, and its product is involved ingermination of spores. J Bacteriol 1999, 181:4584-4591.

74. Moriyama R, Fukuoka H, Miyata S, Kudoh S, Hattori A, Kozuka S, Yas-uda Y, Tochikubo K, Makino S: Expression of a germination-spe-cific amidase, SleB, of bacilli in the forespore compartmentof sporulating cells and its localization on the exterior side ofthe cortex in dormant spores. J Bacteriol 1999, 181:2373-2378.

75. Setlow P: Mechanisms which contribute to the long-term sur-vival of spores of Bacillus species. Soc Appl Bacteriol Symp Ser1994, 23:49S-60S.

76. Bourne N, FitzJames PC, Aronson AI: Structural and germinationdefects of Bacillus subtilis spores with altered contents of aspore coat protein. J Bacteriol 1991, 173:6618-6625.

77. Roels S, Driks A, Losick R: Characterization of spoIVA, a sporu-lation gene involved in coat morphogenesis in Bacillussubtilis. J Bacteriol 1992, 174:575-585.

78. Takamatsu H, Imamura A, Kodama T, Asai K, Ogasawara N, WatabeK: The yabG gene of Bacillus subtilis encodes a sporulationspecific protease which is involved in the processing of sev-eral spore coat proteins. FEMS Microbiol Lett 2000, 192:33-38.

79. Takamatsu H, Watabe K: Assembly and genetics of spore pro-tective structures. Cell Mol Life Sci 2002, 59:434-444.

80. Driks A: Proteins of the spore core and coat. In Bacillus subtilisand its Closest Relatives: From Genes to Cells Edited by: Sonenshein AL,Hoch JA, Losick R. Washington, DC: American Society forMicrobiology; 2002:527-535.

81. Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, MonsonR, Losick R, Grossman AD: Genome-wide analysis of the sta-tionary-phase sigma factor (sigma-H) regulon of Bacillussubtilis. J Bacteriol 2002, 184:4881-4890.

82. Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, SetlowP, Losick R, Eichenberger P: The forespore line of gene expres-sion in Bacillus subtilis. J Mol Biol 2006, 358:16-37.

83. Eichenberger P, Fujita M, Jensen ST, Conlon EM, Rudner DZ, WangST, Ferguson C, Haga K, Sato T, Liu JS, Losick R: The program ofgene transcription for a single differentiating cell type duringsporulation in Bacillus subtilis. PLoS Biol 2004, 2:e328.

Genome Biology 2008, 9:R114